Littérature scientifique sur le sujet « Rejection antibody-mediated »

Although the short-term outcomes of solid allograft survival have improved substantially over the last few decades, there has been no significant improvement in long-term survival of solid allografts. This thesis presents the initial characterisation of alloantibody mediated rejection in a murine heart transplant model, with particular focus on the impact of the different phases of the humoral alloimmune response (follicular or germinal centre) on graft rejection.

Immune mediated injury is a major cause of late kidney graft loss. Donor specific HLA antibodies are widely believed to cause chronic rejection because their presence correlates strongly with worse graft outcome. However, some renal transplant recipients have circulating DSA for many years whilst maintaining stable graft function. This dichotomy has led to the hypothesis that the cause of the immune mediated damage observed in chronic rejection is likely to be multi-factorial. It has previously been demonstrated that donor specific CD4+ T cell activation can be observed in patients with chronic antibody mediated rejection using an IFNγ ELISpot assay. In some samples, this cellular activation was dependent on the presence of B cells, and in others the cellular activation appeared to be regulated by the presence of B cells. In this thesis, the same patterns of reactivity were observed in renal transplant recipients with chronic antibody mediated rejection recruited onto the RituxiCAN C4 clinical trial. HLA binding B cells were detected flow cytometrically and associations between their phenotype and the different patterns of reactivity were analysed. The majority of HLA binding B cells were found to be IgM+ and were underrepresented in the class switched memory B cell population (CD27+IgM-/CD45RBmem55+IgM-) relative to the global B cell population. B cell dependent IFNγ production in response to donor specific HLA was associated with a higher ratio of IgM+ memory to IgM+ naïve B cells in the HLA binding B cell population. CMV gB was used as a model protein to investigate the individual contribution that memory (CD27+) and naïve (CD27-) B cells make to antigen specific IFNγ production. The results suggest that memory B cells support IFNγ production and naïve B cells may be suppressing IFNγ production. Collectively the results in this thesis support a role for a cell-mediated component in chronic antibody mediated rejection. In order to develop more effective treatments for chronic rejection, further investigation into the contribution that T and B cells make to this process is warranted.

INTRODUCTION Heart transplant still represent the most important therapeutic strategy for end-stage cardiomyopathies. Humoral rejection was recognized as a distinct clinico-pathologic entity characterized by acute allograft rejection associated with the production of antidonor HLA antibodies and poor prognosis. For the first time on renal biopsy from patients with kidney function deterioration and absence of cellular rejection C4d was identified as an important diagnostic biomarker for antibody mediated rejection and was strongly correlated with the presence of circulating anti-HLA antibodies. In heart transplantation in the ISHLT working formulation of 2004 the diagnostic criteria of AMR were defined only as present or absent , recognizing grade 0 without histological and immunopathological signs of humoral rejection and grade 1 in the setting of both histological and immunopathological signs of humoral rejection. However the role of AMR remained controversial with different reported incidence between different centers. Even though we were able to obtain a better control of cellular rejection with a decreasing rate of positive Acute Cellular Rejection (ACR) requiring drug therapy modification, no concomitantly reduction in graft loss for cardiac allograft vasculopathy was observed. Recently the attention was mainly focused on humoral rejection and on our ability to detect it through immunohistopathological markers. The aims of the present thesis were summarized in three major objectives: 1) to study the pathophysiology of Antibody Mediated Rejection (AMR) in heart transplant recipients; 2) to assess the diagnostic ability of pathological features to identify AMR on monitoring post-transplant endomyocardial biopsies (EMBs); 3) to clarify the prognosis of AMR in the early and late post-transplant period. MATERIALS AND METHODS. As C4d staining has been performed on a routine basis in heart transplant recipients at the University of Padua's Heart Transplantation Center since 2005, 1.452 consecutive EMBs from 131 patients (mean age 48.9±18.3) were reviewed. Donor mean age was 41,6±15,2 years and 15% had more than 60 year old. Adult population. To study survival in patients with C4d positivity on EMBs we evaluated 985 consecutive biopsies from a total of 107 adult patients (n=85, 79% of these were males) with a median age at the time of transplantation of 55 years (range 17-73 years). Our study population was divided into 4 groups on the basis of the patients’ C4d, DSA, and graft function profile: - C4d positive, DSA negative, without graft dysfunction (asymptomatic AMR) = group 1; - C4d positive, DSA positive, without graft dysfunction (asymptomatic AMR) = group 2; - C4d positive, DSA positive, presence of graft dysfunction (symptomatic AMR) = group 3 - C4d negative, DSA negative, without graft dysfunction, control group = group 0. Pediatric population. We evaluated 226 consecutive biopsies from 24 transplant patients (mean age 8,1±6,3 years). The immunoperoxidase staining was applied routinely both in paediatric and adult patients. The staining for C4d was performed retrospectively using affinity-purified antihuman C4d rabbit polyclonal antibody. The grading system was standardized and was defined in four grades: grade 0 = negative; grade 1 = weak staining with focal distribution, grade 2 = moderate staining with multifocal distribution; grade 3 = strong staining with diffuse pattern. We considered positive grades 2 and 3.The cut off value between grade 1 and grade 2 was 50% of intramyocardial capillaries involved. Immunostaining for C3d, was performed on paraffin embedded section and using a polyclonal antibody C3d.The grading system for C3d immunostaining was the same as for C4d. Histological features of AMR on EMBs. Of the 1452 EMBs, 87 were positive for C4d staining (6% 87/1452) from 61 patients, 26 EMBs from 13 patients had repeated C4d positivity. Sixty-six patients staining negative for C4d were matched for pre-transplant diagnosis, time after transplantation age, and acute cellular rejection (ACR) grading (case-control study). Among the 61 C4d positive patients 9 were diagnosed as AMR. In this case-control study we also applied C3d staining. Two pathologists reviewed blindly all morphological features associated to humoral damage: endothelial swelling, interstitial edema, intracapillary mononuclear cells, intracapillary neutrophils, myocite damage, myocite necrosis, hemorrhage, intravascular microthrombi according to ISHLT 2005 classification. Donor specific Antibodies (DSA) Assessment. Since the study of C4d has been mainly retrospective only in a subgroup of patients circulating anti HLA antibodies could be performed. In thirty-seven patient circulating Anti HLA antibodies were assessed and resulted 26 positive and 9 negative. IgG anti-HLA reactivity in the sera, obtain before transplantation and at the time of C4d positive detection on EMBs, was analyzed using bead-based screening assays, referred to as Luminex methodology. All sera tested positive at screening, were retested with Single Antigen beads in order to determine the antibody specificity. Contrast-enhanced transthoracic Doppler echocardiography. We selected 19 patients who at the time of biopsy stained positive for C4d and in whom echocardiography was performed for Coronary Flow Reserve (CFR) evaluation using CE-TTE before and after adenosine infusion. CFR was calculated as ratio of peak diastolic velocity(hyperemia) and peak diastolic velocity (basal). Statistical analysis. . The comparison between C4d groups was conducted with the Fisher’s exact test in case of categorical variables and the Kruskal-Wallis test for quantitative variables. The Kaplan-Meier method was applied to estimate the C4d groups’ survival functions and the log–rank test was used to compare survival between groups. The Cox’s regression model was used to estimate unadjusted and sex and age adjusted hazard ratios (HR). Sensitivity and specificity, were reported with 95% confidence interval calculated with the exact method RESULTS Clinic-pathological profile of adult population The 22 patients in group 1 (61% of total C4d+ve) showed a 18 fold higher mortality risk compared to the C4d negative patients (95% CI 1.960 to 160.022). The 6 patients in group 2 had a 61 fold higher mortality risk (95% CI 3.399 to 1110.360). The 8 patients in group 3 had a 32 fold higher mortality risk (95% CI 5.884 to 179.432), overall p<0.0001 Overall the C4d positive patients showed a statistically significant reduction in survival compared to the C4d negative patients and this observation was maintained in the three different groups When the asymptomatic (groups 1 and 2) and symptomatic patients (group 3) were compared with the control group an 18 and 26 fold increase mortality risk was observed, respectively. Clinic-pathological profile of pediatric population. Seven patients (33%) showed a C4d +ve intra-graft capillary deposition. Of these 4 were positive for circulating donor specific anti HLA antibodies; none were positive in the C4d negative group. One patient presented also graft dysfunction (14% of C4d+ve) within 3 months after transplantation. One patient with C4d positivity died for heart failure after 2 years since C4d positivity detection. The other five patients with C4d positivity are still alive at 2, 3, 4 , 14 years respectively. In the C4d positive group the rejection score was higher compare to C4d negative patients. (2.2 vs 0.2, p<0,05) Morphological parameters on Endomyocardial biopsy (EMB) to detect Asymptomatic AMR (AsAMR) and Symptomatic AMR (AMR). Of the 8 histological characteristic evaluated on Hematoxilyn Eosin , only two (endothelial swelling and interstitial edema) could be considered fair predictors of C4d capillary positivity in the light of their sensitivity: endothelial swelling with a 78.7 % sensitivity (its specificity was very poor 28.8%) and interstitial edema with a 77.1% sensitivity (its specificity was 31.8%). Intracapillary macrophages had a sensitivity of 39.3% only and a specificity of 68.2%. The sensitivity and specificity of the histological parameters in EMBs of patients with C4d positivity (both asymptomatic and symptomatic) were similar to those observed in EMBs of patients affected by symptomatic AMR ROC curve combining endothelial swelling and intra-capillary aggregates of macrophages did not improve the capacity to predict presence of circulating anti HLA antibodies. Immunostaining for C3d in the diagnosis of AMR. In the asymptomatic AMR group (52 EMBs of 52 patients) 31 were also positive for C3d (59,6%, 31/52). In the symptomatic AMR group (9 EMBs of 9 patients) 5 were also positive for C3d (55% 5/9). The sensitivity of C3d to predicted DSA was 42.3% and the specificity of C3d was 56% for DSA. Combining C4d and C3d to predicting circulating DSA did not increase the sensitivity and specificity of C4d. Alloantibody profile in symptomatic AMR. Of the 9 symptomatic AMR patients, 5 had been followed up for C4d positivity on EMBs and circulating DSA. The results showed that MFI for class I and class II changed according to the treatment. High level of MFI (>10000) strongly correlated with diffuse and strong intensity of C4d staining; with MFI ranging between 1000-9000 C4d was variably detected; with MFI < 1000 C4d was always negative. Microvasculopathy and C4d. Seven out of 19 patients, in whom we performed echostress(or where ECHO stress was reduced) had C4d positive immunostaining while 12 were negative (average age 50.1±8.7 years). In the group of patients with C4d positivity 4 were late symptomatic AMR (mean time after Htx 13.9±4.9 years) and three of them died after few months since the diagnosis of AMR. Three patients had asymptomatic AMR (mean time after HTx 5.3±3.4 years). Coronary flow reserve (CFR) of patients with C4d+ was statistically significant reduced compared to C4d- group (1.28±0.48 vs 3.28±1.03 respectively, p=0.0297) suggesting a relation between humoral rejection and microvascular damage. Even in pts without CAV at angiography C4d positivity identified a low value of CFR in the C4d+ group (p=0.0502). CONCLUSIONS. Our findings indicate that C4d+ve is an important marker for diagnosis of AMR , thus identifying asymptomatic AMR . C4d predicts worse prognosis and DSA and graft dysfunction further improve risk stratification. C4d+ve and DSA can be used as early mortality predictors in patients without signs of graft dysfunction. AMR is a complex and ongoing phenomenon with different phenotypic features. Morphological parameters alone, are not adequately sensitive and carry low positive likelihood ratio as screening tools for early asymptomatic AMR detection. Screening recommendations has been recently modified to include more sensitive tests such as C4d staining in the routine protocol to improve patient risk stratification. The assessment of anti HLA antibodies, class I and II and their relation with C4d (complement activation)showed : 1) a difference in type of anti HLA-DSA in the early and late period post-transplant with class I anti HLA early appearance and class II anti HLA late onset after transplant 2) high levels of MFI correlate with C4d deposition on EMBs. Endothelial damage and complement deposition produce microvascular remodeling leading to microvasculopathy with increased graft loss.
INTRODUZIONE Il trapianto cardiaco rappresenta ancora la più importante strategia terapeutica nei pezienti con scompenso cardiaco refrattario alla terapia medica. Nell’ultimo decennio la attenzione clinica è stata rivolta al rigetto umorale in seguito al dato che la percentuale di perdita del garft per rigetto cronico non è cambiata, nonostante la terapia immunosoppressiva controlli in modo ottimale il rigetto cellulo-mediato. Il rigetto umorale viene definito come una entità clinico-patologica caratterizzata dalla presenza di anticorpi anti HLA (complesso maggiore di istocompatibilità) circolanti che provocano danno endoteliale sull’organo trapiantato con conseguente impatto sulla mortalità. Per la prima volta in biopsie renali di pazienti con disfunzione del graft in assenza di rigetto cellulo-mediato, è stato evidenziato deposito di C4d sulla superficie endoteliale, sinonimo di attivazione del complemento , strettamente correlato con la presenza di anticorpi anti HLA circolanti nel siero. Le linee guida internazionali che definiscono i criteri patologici per la diagnostica del rigetto nel trapianto di cuore, solo nel 2004 per la prima volta definiscono il rigetto umorale come entità patologica distinta indicandone i criteri diagnostici istologici e immunopatologici maggiori, riconoscendo come grado 0 assenza di segni istologici e immunopatologici e grado 1 la presenza di segni istologici e immunopatologici di rigetto umorale. Nell’ultima decade la letteratura internazione ha rivolto la propria attenzione alla ricerca del significato prognostico del rigetto umorale in relazione al rigetto cronico e alla ricerca di nuovi biomarcatori per la diagnosi precoce del rigetto umorale. Gli obiettivi della presente tesi di dottorato possono essere riassunti in tre principali puntii: 1) studio della fisiopatologia del rigetto umorale o rigetto anticorpo mediato nel trapianto di cuore; 2) valutazione della capacità diagnostica dei criteri istopatologici definiti dalle linee guida internazioni del 2004, su biopsie endomiocardiche di monitoraggio post-trapainto; 3) definizione del significato prognostico del rigetto anticorpo mediato sia nella fase precoce che tardiva post-trapianto. MATERIALI E METODI. Dal 2005, nel nostro centro di riferimento per il trapianto cardiaco dell’Università di Padova, abbiamo studiato routinariamente il biomarcatore di attivazione del complemento (C4d) con tecnica immunoistotchimica su tessuto (biopsie endomiocardiche di monitoraggio). Abbiamo valutato 1452 biopsie endomiocardiche (BEM) da 131 pazienti (età media 48.9±18.3). Popolazione adulta. Per studiare la sopravvivenza di pazienti adulti che presentano una positività al C4d nel loro follow up, abbiamo studiato 985 BEM di 107 pazienti (79% maschi) con età al trapianto di 55 anni (mediana) range 17-73 anni. La nostra popolazione adulta è stata suddivisa il quattro gruppi sulla base del profilo del C4d nelle BEM, degli anticorpi circolanti anti HLA donatore specifici (DSA), e della disfunzione del graft (riduzione della frazione di eiezione, anomalie elettrocardiogarfiche): • C4d positivo, DSA negativi, assenza di disfunzione del garft (rigetto anticorpo-mediato asintomatico)= gruppo 1 • C4d positivo, DSA positivi, assenza di disfunzione del graft (rigetto anticorpo-mediato asintomatico)= gruppo 2 • C4d positivo, DSA positivi, presenza di segni clinici di disfunzione del graft (rigetto anticorpo-mediato sintomatico)= gruppo 3. • C4d negativo, DSA negativi, assenza di disfunzione clinica del graft (gruppo controllo )= gruppo 0. Popolazione pediatrica. Abbiamo valutato 226 biopsie provenienti da 24 pazienti trapiantati (età media 8,1±6,3 anni). Tecnica immunoistochimica è stata applicata routinariamente sia nella popolazione adulta sia nella popolazione pediatrica. La tecnica immunoistochimica è stata applicata sia retrospetticamente che prospetticamente usando anticorpo policlonale per C4d. Il grading per la valutazione della colorazione immunoistochimica è stata standardizzato in quattro gradi: grado 0= negativo; grado 1= debole intensità, focale coinvolgimento dei capillari; grado 2= moderata intensità e coinvolgimento multifocale dei capillari e grado 3= colorazione intensa e coinvolgimento diffuso dei capillari. Si considera positivo grado 2 e grado 3. E’ stata inoltre eseguita l’immunoistochimica per C3d su materiale paraffinato applicando lo stesso schema di valutazione del C4d. Valutazione dei criteri diagnostici morfologici di rigetto anticorpo mediato. Delle 1452 BEM valutate, 87 erano positive per il C4d (6% 87/1452) in 61 pazienti, 26 BEM di 13 pazienti hanno mostrato un C4d positivo in più di un biopsia. Sessantasei pazienti negativi per il C4d sono stati selezionati e confrontati secondo i seguenti criteri: diagnosi pre-trapianto, età dopo il trapianto, grading del rigetto cellulo-mediato (studio caso-controllo). Tra i 61 pazienti positivi per il C4d 9 avevano rigetto anticorpo-mediato sintomatico, In questo studio caso-controllo abbiamo studiato anche il C3d. Due Patologi hanno rivisto “blindly” tutti i criteri istologici associati al rigetto umorale: edema endoteliale, edema interstiziale, cellule mononucleari intracapillari, neutrofili intracapillari, danno miocitario, necrosi miocitaria, emorragia, microtrombi intravascolari in accordo con le linee guida internazionali “ISHLT working formulation “ del 2004. Determinazione degli anticorpi anti HLA donatore specifici. La valutazione degli anticorpi circolanti è stata eseguita su 37 casi, in virtù del fatto che il nostro studio è stata condotto anche retrospetticamente. E’ stata utilizzata la tecnica Luminex per testare la reattività degli anticorpi anti HLA tipo IgG con tecnica “bead-based screening assay” da sieri ottenuti pre-trapianto e post-trapianto, quest’ultimo in concomitanza con la positività del C4d nelle BEM. Inoltre in una sottopopolazione di pazienti con rigetto umorale sintomatico gli anticorpi circolanti sono stati quantificati misurando la media di intensità di fluorescenza (MFI). Valutazione della riserva coronarica tramite tecnica ecocardiografica non invasiva. Abbiamo selezionato 19 pazienti in cui al momento della biopsia endomiocardica di monitoraggio post-trapianto è stata eseguita ecocardiograficamente.La Riserva Coronarica valutata prima e dopo l’infusione di adenosina La riserva coronarica (CFR) è stata calcolata con rapporto tra picco di velocità diastolica (iperemia) e picco di velocità diastolica (basale). Analisi statistica. Il confronto tra i vari gruppi C4d è stato calcolato con test Fisher nei caso di variabili categoriali e con test di Kruskal-Wallis per variabili quantitative. E’ stato applicato il metodo di Kaplan-Meyer per studiare la sopravvivenza dei vari gruppi positivi per C4d. Test di regressione di Cox per stimare Hazard ratio. Sono state calcolate sensibilità e specificità dei vari parametri morfologici e riportate con intervallo di confidenza del 95%. RISULTATI. Profilo clinico-patologico della popolazione adulta. Ventidue pazienti del gruppo 1 (61% del totale dei C4d positivi) mostano un rischio di mortalità 18 volte maggiore rispetto al gruppo di controllo (95% CI 1,960 – 160,022).. I sei pazienti del gruppo 2 hanno un rischio di mortalità 61 volte maggiore rispetto al gruppo di controllo (95% CI 3.399-1110.360). Gli 8 pazienti del gruppo 3 hanno un rischio di mortalità 32 volte maggiore rispetto al gruppo di controllo con una p<0,0001. Se si considerano nella totalità i pazienti C4d positivi mostrano una riduzione statisticamente significativa della sopravvivenza rispetto al gruppo C4d negativo. Si osserva un aumento del rischio di mortalità sia nei gruppi 1 e 2 (asintomatici) che nel gruppo 3 sintomatico Profilo clinico-patologico della popolazione pediatrica. Sette pazienti (33%) mostrano una positività al C4d nelle BEM di monitoraggio. Di questi 4 presentavano anticorpi anti HLA circolanti; nessuno era positivo nel gruppo di controllo. Un paziente con positività al C4d ha presentato disfunzione del graft (14% dei C4d+) entro i 3 mesi dal trapianto. Un paziente C4d positivo è morto per scompenso cardiaco dopo circa 2 anni dalla comparsa della positività del C4d nelle BEM. Gli altri cinque pazienti con C4d + sono attualmente vivi a rispettivamente 2,3,4,14 anni di follow up. Nel gruppo C4d+ il “rejection score” per rigetto cellulare è maggiore rispetto al gruppo di controllo (2,2 vs 0,2, p<0,05). Parametri morfologici per la diagnosi di rigetto anticorpo-mediato sintomatico e asintomatico nelle biopsie endomiocardiche di monitoraggio. Degli 8 criteri morfologici valutati all’ematosillina ed eosina, solo due (edema endoteliale ed interstiziale) possono essere considerati predittivi di positività capillare al C4d. la sensibilità dell’edema endoteliale è del 78,8% e la sensibilità dell’edema interstiziale è del 77,1%. Accumulo intracapillare di cellule mononucleari hanno una sensibilità del 39,3% nel predire la positività al C4d. La curva ROC mostra come i due criteri valutati assieme e riconosciuti essere i più importanti associati al danno anticorpo-mediato non aumentano la capacità di predite la presenza di anticorpi circolanti anti HLA. Immunoistotchimica per C3d nella diagnostica del rigetto umorale. Nel gruppo degli asintomatici (52 BEM di 52 pazienti) 31 BEM sono positive anche per C3d (59,6%, 31/54). Nel gruppo dei sintomatici (9 BEM per 9 pazienti) 5 BEM erano positivi al C3d (55% 5/9). La sensibilità del C3d nel predire la presenza di anticorpi anti HLA circolanti è del 42,3% e la specificità del 56%. Combinando C4d e C3d la sensibilità e specificità nel predire la presenza di anticorpi circolanti non aumenta. Profilo anticorpale nei pazienti con rigetto anticorpo mediato sitomatico. Dei 9 pazienti sintomatici della nostra popolazione studiata, 5 sono stati seguiti in follow up sia per C4d sia per anticorpi circolanti. I risultati mostrano che la quantità di anticorpo circolante (MFI) cambia in relazione al trattamento. Inoltre alti livelli di MFI >10000 correlano strettamente con grado 3 del C4d (diffuso coinvolgimento capillare con colorazione intesa), con MFI tra 1000-9000 il C4d ha una intensità ed una distribuzione variabile.; con MFI <1000 C4d è sempre negativo. C4d e microvasculopatia. La riserva coronarica di pazienti con C4d + è ridotta rispetto al gruppo di controllo rappresentato da pazienti con C4d- (1,28±0,48 vs 3,28±1,03 rispettivamente, p=0,0297) suggerendo una relazione tra il rigetto anticorpo mediato e il danno microvascolare. Anche nei pazienti senza coronaropatia del graft i pazienti con C4d+ mostrano una riserva coronarica inferiore rispetto al gruppo di controllo (C4d-) (p=0,0502). CONCLUSIONI I nostri risultati dimostrano come il C4d sia un indicatore oltre che diagnostico anche prognostico per rigetto umorale e la sua positività è sinonimo di prognosi negativa anche in assenza di sintomi clinici . La valutazione di C4d, anticorpi circolanti e disfunzione del graft ci permette di stratificare il rischio di mortalità. I parametri morfologici da soli non sono sufficientemente sensibili e specifici per la diagnostica del rigetto umorale e dunque non sono adeguati per una valutazione di screening. Alla luce dei nostri risultati raccomandiamo il C4d come test di screening per individuare pazienti asintomatici. Lo studio degli anticorpi circolanti hanno mostrato che alti livelli di anticorpi correlano con la positività capillare del C4d . Il danno endoteliale e il deposito di complemento determinano rimodellamento vascolare che porta a lungo termine

This thesis tests the hypothesis that Tregs with indirect allospecificity can prevent humoral rejection. Anti-CD4 monoclonal antibody and PVG.R8 donor-specific blood transfusion (DST) given pre-transplant led to long-term PVG.R8 heart graft survival in two-thirds of PVG.RT1^u recipients. Alloantibody production was abrogated in these animals and tolerance was allospecific. Histology of long-term surviving grafts showed no evidence of chronic rejection. Naïve syngeneic spenocytes injected into tolerant PVG.RT1^u animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4+ T cell-reconstituted congenitally athymic PVG.RT1^u rats confirmed that regulation in tolerant animals was mediated by spenic CD4+ T cells. These CD4+ Tregs were allospecific. Other mechanisms of tolerance in this model were explored by breaking regulation in tolerant rats, either by immunising with immunodominant linear allopeptide, or by depleting tolerant CD4+ T cells with anti-CD4 monoclonal antibody. Surprisingly, this resulted in neither alloantibody generation nor graft rejection, suggesting that tolerance also resides within alloantigen-specific B cells. These results demonstrated that anti-CD4 + DST treatments results in the development of CD4+ Tregs that recognise alloantigen via the indirect pathway and act in an antigen-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell component that persists after CD4+ T cell help has been made available. These findings may be of use in designing clinically-applicable tolerogenic strategies in the future.

We determined in a rat model (1) the presence and dynamics of alloantibodies recognizing MHC complexes on quiescent Brown-Norway (BN) splenic cells in the sera of Lewis (LEW) recipients of Brown-Norway iliolumbar vein grafts under tacrolimus immunosuppression; and (2) the presence of immunoglobulins in the wall of acute rejected vein allografts.

En transplantation d’organes solides, la principale cause de perte de greffons à long terme est le rejet humoral. Le rejet humoral est une maladie de la vasculature du greffon, caractérisé par une inflammation et une forte concentration sérique des anticorps dirigés contre le greffon. Les études récentes ont souligné la forte prévalence des anticorps spécifiques à HLA-DQ parmi des anticorps spécifiques du donneur formés post-transplantation. Cette thèse explore l’importance de l’expression de HLA-DQ par les cellules endothéliales dans la pathogenèse du rejet humoral. Afin d’étudier l’expression de HLA-DQ, des cellules endothéliales microvasculaires ont été stimulées avec des cytokines pro-inflammatoires. L’inflammation induite altère l’immunogénicité des cellules endothéliales et donc modifie la qualité des synapses immunologiques avec les lymphocytes T CD4+ alloréactifs. Ainsi, l’inflammation chronique a diminué la capacité des cellules endothéliales à induire l’expansion des lymphocytes T CD4+ régulateurs FoxP3high (Treg). De plus, la fixation des anticorps anti-HLA-DQ aux cellules endothéliales, en synergie avec l’inflammation, réduit davantage l’expansion des lymphocytes T CD4+ régulateurs. Cette baisse des cellules anti-inflammatoires peut altérer la tolérance des allogreffes, promouvoir les réponses pro-inflammatoires et aggraver le rejet
In solid organ transplantation, antibody-mediated rejection is currently the major cause of allograft failure. Antibody-mediated rejection is a disease of the allograft vasculature, characterised by inflammation and circulating donor-specific antibodies. Recent studies have exposed the particularly high prevalence of antibodies targeting HLA-DQ amongst de novo donor-specific antibodies produced post-transplantation. This thesis explores the significance of endothelial HLA-DQ expression in the pathogenesis of antibody-mediated rejection. HLA-DQ expression in in vitro microvascular endothelial cell cultures required sustained exposure to inflammatory cytokines. Such inflammation modified endothelial immunogenicity and subsequent synapses with alloreactive CD4+ T lymphocytes. As a consequence, sustained inflammation compromised the capacity of endothelial cells to expand anti-inflammatory regulatory T cells. Moreover, the binding of HLA-DQ alloantibodies to the endothelial cells synergised with inflammation to further diminish regulatory T cell expansion. This reduction in anti-inflammatory cells may impair allograft tolerance, promote proinflammatory responses and exacerbate rejection

Background: Rejection is considered as a major barrier to achieve successful transplantation. Non self-human leucocyte antigen (HLA) is a well-known antigenic target for antibodies binding that can result in antibody-mediated rejection (AMR). To reduce risk of rejection in kidney transplant, preventive measures are undertaken, which include HLA-matching between donor and recipient, and in-vitro pre-transplant crossmatch with potential donor cells and recipient sera, furthermore, periodic HLA antibodies monitoring for donor-specific antibodies (DSA) is carried out before and after transplant. Nevertheless, allograft may still fail despite the above measures, which suggests other antigens besides HLA can also contribute to renal rejection. In fact, polymorphic major histocompatibility complex (MHC) class I–related chain A (MICA) antigens and Angiotensin II type 1 receptor (AT1R) antigens have been reported as likely targets in AMR. However, the effect of non-HLA antibodies such as anti-MICA and anti-AT1R antibodies in rejection are not fully defined. This implies there is an imminent need to elucidate the role of non-HLA antibodies in allograft AMR cases which are not mediated by HLA antibodies. Aim: To retrospectively evaluate the occurrence of MICA and AT1R antibodies in 21 clinical AMR cases without detectable HLA antibodies or HLA antibodies that were not target against donor HLA. Methods: Twenty-one cases with suspected non-HLA mediated post-transplant rejection were retrieved. Eplet analysis was utilized to confirm that the detectable HLA-DR antibodies in one of the samples were not cross-reactive towards a donor’s antigen. Sera from 21 non-AMR cases were used as controls. All sera were subjected to MICA antibody and AT1R antibody screening. Identified positive cases were further examined with their pre-transplant sera to assess whether the AT1R and/or MICA antibodies were already pre-formed before transplantation. The sensitization histories of transfusion, pregnancy and previous transplantation were recorded. Results: Nine of twenty-one cases were detected with MICA and/or AT1R antibodies. 7 samples were detected with MICA antibodies while 3 samples were detected with AT1R antibodies. A sample was detected with both MICA and AT1R antibodies. Importantly, the presence of MICA/AT1R antibodies appeared to be strongly associated with rejection caused by non-HLA antigens (p=0.0007). All controlled cases were found to be negative for MICA and AT1R antibodies. Pre-transplant sera of the positive cases were further screened and pre-formed antibodies were detected in 3 of the positive MICA cases, and 1 of the positive AT1R cases. Since no AT1R and MICA genotyping of the donor was carried out previously, it was uncertain that the allograft rejection was induced by the donor specific pre-formed antibodies generated in the pre-transplant sensitization events. Nonetheless, AT1R and MICA antibodies appeared to be induced by the allograft in the remaining 5 cases. Conclusion: Presence of MICA/AT1R antibodies appeared to be associated with the investigated AMR cases without detectable HLA antibodies. Some evidence suggested the production of these non-HLA antibodies could be induced by transfusion sensitization or allograft upon transplantation.
published_or_final_version
Pathology
Master
Master of Medical Sciences

Rejection of the transplanted kidney is an important cause of graft loss despite modern cross-matching techniques and immunosuppressive agents. The incidence of acute rejection episodes in the first post-transplant year is down to less than 15% in low-risk recipients, but as many as one-third of allograft losses over 10 years result from alloimmunity. Rejection may occur at any time following transplantation, from minutes—hyperacute, to days—acute, or in the longer term—chronic. Rejection can be predominantly through either T-cell-mediated or antibody-mediated mechanisms. It may present clinically as either abrupt or insidious dysfunction of the graft, or it may be subclinical and thus silent, detected only by protocol biopsy or other technology. The prevention and treatment of T-cell-mediated rejection is usually successful with current immunosuppressive agents. Antibody-mediated rejection, on the other hand, is not easily treated and is the principal cause of late renal allograft loss. This chapter presents the concepts and details of this central issue in clinical transplantation.

Abstract Long-Term Care of Kidney Transplant Patients with expert authors around the globe provides details on topics relevant to kidney transplant including current survival rates, phases following kidney transplantation, transplant wellness, current immunosuppressive management, alloimmune injuries such as early and late T-cell and antibody-mediated rejections as well as various non-alloimmune factors such as post-transplant infections, cardio-vascular diseases, cancers are discussed in special chapters. Comprehensive chapters were dedicated to psychological and social factors influencing kidney transplant followed by special chapters on pediatric kidney transplantation and multiorgan transplantation. The final chapters on “Transplant Tolerance” and the “Conclusion” summarizes all chapters and highlights potential novel advances that might become game changer in kidney transplant field. This comprehensive book becomes a desk reference, provides better means to care for patients and benefits all readers all over the world who are caring for kidney transplant patients.